Project summary Cytosolic DNA infectious microbe triggers the DNA sensor, cyclic GMP-AMP synthase (cGAS), which elicits interferon production signal cascades. Normally host DNA is sequestered in the nucleus and mitochondria, thereby preventing unwanted interferon (IFN) activation. However, cellular stress caused by infection and pathophysiological conditions leads to the leakage of mitochondrial DNA (mtDNA) into cytoplasm and elicits DNA-mediate innate immune response. The aberrant activation by DNA sensing pathway can result in inflammatory and autoimmune diseases, such as systemic lupus erythematosus. However, how host limits excessive or detrimental immune responses to viral DNA and mtDNA is not well elucidated. Thus, it is pressing to define the protective mechanisms that prevent aberrant activation of cGAS-mediated innate immunity. This application is to elucidate a host protection mechanism by which complement C1q binding protein (C1QBP) prevents cGAS from over-activating. Aim 1 will define the role of C1QBP in cytosolic DNA-mediated innate immunity in vitro and in vivo. We will define the inhibitory role of C1QBP in cGAS activation by overexpression and CRISPR knockout in macrophage cells. We will also use knockout mice to define the in vivo role of C1QBP in cGAS-mediated innate immunity. Aim 2 will determine the protective mechanisms by which C1QBP limits cytosolic DNA-induced cGAS activation. We will examine several hypotheses of how C1QBP inhibits cGAS activity. cGAS activity must be tightly regulated because sustained IFN production can lead to autoimmune diseases. This exploratory R21 application proposes C1QBP as a hidden host protector in mitochondria matrix. When mitochondria are damaged by infection and other cellular stresses, C1QBP is released from mitochondria to inhibit cGAS activity, thereby preventing excessive IFN response triggered by the leaked mtDNA. This study will elucidate an elegant host self-regulation mechanism, providing foundations for developing novel therapeutic strategies for infectious and autoimmune diseases.